ABSTRACT
Based on the formulation method the dry powder inhalers (DPIs) can be divided in too types: carrier-based and carrier-free drug delivery systems. The newest researches report about several high potency carrier-free formulations, where the active ingredient and the excipients are together formulated to the DPI form. However, in Hungary the commercially available DPIs are carrier-based (e.g. lactose), which means that only the mic-onized active ingredient reaches the deeper lungs, the big carrier deposits in the upper airways. The present work is about formulating a high efficacy mannitol-based Pulmonary Drug Delivery System (PDDS), which is able to delivery different types of active ingredients to the deeper lungs with higher deposition rate. The present study involves the physico-chemical and aerodynamical characterisation of mannitol-based PDDS. The results demonstrated the use of the appropriate excipients (leucine, poly-vinyl-alcohol, cyclodextrine) and solvent combination (ethanol-water) during the co-spray drying, increases the inhalation properties of the mannitol. Such carrier systems with optimized properties can increase the aerolization efficacy of the active ingredient.
Subject(s)
Chemistry, Pharmaceutical/methods , Drug Carriers , Drug Delivery Systems/instrumentation , Dry Powder Inhalers , Mannitol/chemistry , Pharmaceutical Preparations/chemistry , Administration, Inhalation , Drug Compounding , Equipment Design , Excipients/chemistry , Humans , Mannitol/administration & dosage , Pharmaceutical Preparations/administration & dosageABSTRACT
The works of Jan Evangelista Purkyne, Gabriel Valentin and Robert Remak showed that the nervous system contains not only nerve fibers, but also cellular elements. The use of microscopes and new fixation techniques have enabled the retrieval of accurate data on the structure of nervous tissue and in many European universities microscopes began to be widely used for histological and morphological studies. The present review summarizes the discoveries of the structure of predominantly vertebrate nerve tissue during the period from 1838 to 1865, made by prominent scholars who described the structure of fibers and cells of the nervous system and demonstrated that some nerve fibers are enwrapped by a sheath. In addition, the first attempts were made to make a cytoarchitectonic description of the spinal cord and brain. During the same time the concept of a neuroglial tissue was introduced, first as a tissue for "gluing" nerve fibers, cells and blood capillaries into one unit, but later some glial cells were described for the first time. Microscopic techniques started to be used for examination of physiological as well as pathological nerve tissues. The overall state of knowledge was just a step away from the emergence of the concept of neurons and glial cells.
Subject(s)
Cell Biology/history , Nervous System/anatomy & histology , Nervous System/cytology , Physiology/history , History, 18th Century , History, 19th Century , HumansABSTRACT
Efforts were already made in antiquity to understand the structure and function of the brain and nervous tissue, but an important impulse for the development of neuroanatomy as a field was the invention of the microscope at the beginning of the 17th century. A brief overview of the history of research of the brain and nerve structure from Marcello Malpighi to Christian Berres shows by what small steps and with what difficulties the investigation of the composition of the nervous tissue advanced. The study of historical sources reveals that despite constantly improving microscopic techniques, many prominent researchers obtained inaccurate data and, on the basis of such data, often reached erroneous conclusions. However, these observations and their interpretation and, in addition, the clashing opinions of scholars of that period greatly influenced the work of later researchers in the field of the microscopic structure and function of the nervous tissue.
Subject(s)
Nervous System/anatomy & histology , Neuroanatomy/history , History, 18th Century , History, 19th Century , Humans , Nervous System/cytology , Neuroanatomy/trendsABSTRACT
The treatise "De structura nervorum" by Jirí Procháska was published in 1779 after his appointment as a professor in Prague. This work is remarkable not only for its anatomical and histological findings, but also for its historical introduction, which contains a very detailed bibliographical review of previous knowledge about the structure of the nervous tissue. The treatise "De structura nervorum" has never been translated from the Latin language, but as a historical document about the level of neuroscience research conducted by a famous Czech researcher, it deserves further analysis. The present article includes a historical overview of knowledge about the structure of nervous tissue up to the late 18th century from the perspective of today, a translation of the historical introduction about the medieval knowledge of the structure of the nervous tissue and documenting the way in which Jirí Procháska processed his bibliography, a translation and interpretation of his neurohistological observations and an analysis of the results in the light of current knowledge.
Subject(s)
Nervous System/anatomy & histology , Neuroanatomy/history , Publications/history , Czechoslovakia , History, 18th Century , History, 19th Century , Humans , Nervous System/cytologyABSTRACT
The Czech anatomist and physiologist of the 18th century, Jirí Procháska (1749-1820), ranks among the major figures of Czech cultural history. Due to historical circumstances, the works of Jirí Procháska were published mostly in Latin and only some in German. However, given that only two of his works have been translated into Czech and one of them also partially into English, the results of his extensive research activities are currently unavailable not only to the international scientific community, but also to the Czech scientific community. Although his research reflected the time in which he lived and thus has been re-evaluated by later researchers, his achievements undoubtedly belong to the major intellectual heritage of Czech science and certainly deserve attention as such. It is therefore our duty not only to remember the work and legacy of Jirí Procháska, which significantly influenced the development of our knowledge, but also to try to critically assess his contribution in terms of today. The article surveys the important biographical events of Jirí Procháska's life, taking into account the importance of his research and teaching.
Subject(s)
Anatomy/history , Physiology/history , Czechoslovakia , History, 18th Century , History, 19th Century , HumansABSTRACT
Sonic hedgehog (Shh) and Wnt-7a are morphogens involved in embryonic as well as ongoing adult neurogenesis. Their effects on the differentiation and membrane properties of neonatal neural stem/progenitor cells (NS/PCs) were studied in vitro using NS/PCs transduced with either Shh or Wnt-7a. Eight days after the onset of in vitro differentiation the cells were analyzed for the expression of neuronal and glial markers using immunocytochemical and Western blot analysis, and their membrane properties were characterized using the patch-clamp technique. Our results showed that both Shh and Wnt-7a increased the numbers of cells expressing neuronal markers; however, quantitative immunocytochemical analysis showed that only Wnt-7a enhanced the outgrowth and the development of processes in these cells. In addition, Wnt-7a markedly suppressed gliogenesis. The electrophysiological analysis revealed that Wnt-7a increased, while Shh decreased the incidence of cells displaying a neuron-like current pattern, represented by outwardly rectifying K(+) currents and tetrodotoxin-sensitive Na(+) currents. Additionally, Wnt-7a increased cell proliferation only at the early stages of differentiation, while Shh promoted proliferation within the entire course of differentiation. Thus we can conclude that Shh and Wnt-7a interfere differently with the process of neuronal differentiation and that they promote distinct stages of neuronal differentiation in neonatal NS/PCs.
Subject(s)
Cell Differentiation/genetics , Hedgehog Proteins/physiology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Stem Cells/metabolism , Wnt Proteins/physiology , Animals , Animals, Newborn , Cells, Cultured , Hedgehog Proteins/genetics , Mice , Mice, Inbred C57BL , Neural Stem Cells/cytology , Stem Cells/cytology , Wnt Proteins/geneticsABSTRACT
Astrocytes, the most numerous cells in the brain, weave the canvas of the grey matter and act as the main element of the homoeostatic system of the brain. They shape the microarchitecture of the brain, form neuronal-glial-vascular units, regulate the blood-brain barrier, control microenvironment of the central nervous system and defend nervous system against multitude of insults. Here, we overview the pathological potential of astroglia in various forms of dementias, and hypothesise that both atrophy of astroglia and reactive hypertrophic astrogliosis may develop in parallel during neurodegenerative processes resulting in dementia. We also show that in the transgenic model of Alzheimer's disease, reactive hypertrophic astrocytes surround the neuritic plaques, whereas throughout the brain parenchyma astroglial cells undergo atrophy. Astroglial atrophy may account for early changes in synaptic plasticity and cognitive impairments, which develop before gross neurodegenerative alterations.
Subject(s)
Alzheimer Disease , Astrocytes/metabolism , Astrocytes/pathology , Dementia , Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Animals , Astrocytes/cytology , Dementia/pathology , Dementia/physiopathology , Disease Models, Animal , Glial Fibrillary Acidic Protein/metabolism , Gliosis/metabolism , Gliosis/pathology , Humans , Mice , Mice, TransgenicABSTRACT
The pathological potential of glial cells was recognized already by Rudolf Virchow, Santiago Ramon y Cajal and Pio Del Rio-Ortega. Many functions and roles performed by astroglia in the healthy brain determine their involvement in brain diseases; as indeed any kind of brain insult does affect astrocytes, and their performance in pathological conditions, to a very large extent, determines the survival of the brain parenchyma, the degree of damage and neurological defect. Astrocytes being in general responsible for overall brain homeostasis are involved in virtually every form of brain pathology. Here we provide an overview of recent developments in identifying the role and mechanisms of the pathological potential of astroglia.
Subject(s)
Astrocytes/physiology , Brain Diseases/physiopathology , Animals , Astrocytes/ultrastructure , Brain/pathology , Brain/physiology , Brain/physiopathology , Brain Diseases/pathology , Extracellular Space/chemistry , Gap Junctions/physiology , Gliosis/physiopathology , Humans , Neurons/physiology , Neurotransmitter Agents/metabolism , Potassium/analysis , Potassium/metabolism , Rats , Reactive Oxygen Species/metabolism , Synapses/physiologyABSTRACT
Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.
Subject(s)
Astrocytes/physiology , Neurons/physiology , Stem Cells/physiology , Animals , Astrocytes/cytology , Cell Differentiation , Cell Line, Transformed , Cell Membrane/physiology , Delayed Rectifier Potassium Channels/physiology , Ectoderm/cytology , Gap Junctions/physiology , Immunohistochemistry , Ion Channel Gating , Mice , Neurons/cytology , Patch-Clamp Techniques , Sodium Channels/physiology , Stem Cells/cytology , Tretinoin/pharmacologyABSTRACT
Glutamate release, particularly in pathologic conditions, may result in cellular swelling. The authors studied the effects of glutamate, N-methyl-D-aspartate (NMDA), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on extracellular pH (pH(e)), extracellular potassium concentration ([K(+)](e)), and changes in extracellular space (ECS) diffusion parameters (volume fraction alpha, tortuosity lambda) resulting from cellular swelling. In the isolated spinal cord of 4-to 12-day-old rats, the application of glutamate receptor agonists induced an increase in [K(+)](e), alkaline-acid shifts, a substantial decrease in alpha, and an increase in lambda. After washout of the glutamate receptor agonists, alpha either returned to or overshot normal values, whereas lambda remained elevated. Pretreatment with 20 mmol/L Mg(++), MK801, or CNQX blocked the changes in diffusion parameters, [K(+)](e) and pH(e) evoked by NMDA or AMPA. However, the changes in diffusion parameters also were blocked in Ca(2+)-free solution, which had no effect on the [K(+)](e) increase or acid shift. The authors conclude that increased glutamate release may produce a large, sustained and [Ca(2+)](e)-dependent decrease in alpha and increase in lambda. Repetitive stimulation and pathologic states resulting in glutamate release therefore may lead to changes in ECS volume and tortuosity, affecting volume transmission and enhancing glutamate neurotoxicity and neuronal damage.
Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Glutamic Acid/pharmacology , N-Methylaspartate/pharmacology , Spinal Cord/drug effects , Spinal Cord/pathology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Calcium/pharmacology , Cell Death/drug effects , Chelating Agents/pharmacology , Diffusion , Dizocilpine Maleate/pharmacology , Edema/pathology , Egtazic Acid/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Extracellular Space/drug effects , Extracellular Space/metabolism , Gliosis/metabolism , Gliosis/pathology , Hydrogen-Ion Concentration , In Vitro Techniques , Magnesium/pharmacology , Potassium/metabolism , Rats , Rats, Wistar , Spinal Cord/metabolismABSTRACT
Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.
Subject(s)
Astrocytes/metabolism , Cell Size/physiology , Cortical Spreading Depression/physiology , Glial Fibrillary Acidic Protein/deficiency , Osmotic Pressure/drug effects , Potassium/pharmacology , Animals , Astrocytes/drug effects , Astrocytes/pathology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cell Size/drug effects , Cortical Spreading Depression/drug effects , Diffusion/drug effects , Extracellular Space/drug effects , Extracellular Space/metabolism , Fluorescent Dyes/pharmacokinetics , Glial Fibrillary Acidic Protein/genetics , Intermediate Filaments/drug effects , Intermediate Filaments/metabolism , Intermediate Filaments/pathology , Isoquinolines/pharmacokinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Knockout/anatomy & histology , Mice, Knockout/metabolism , Patch-Clamp Techniques , Potassium/metabolism , Somatosensory Cortex/metabolism , Somatosensory Cortex/physiopathology , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
In rat brain and spinal cord slices, the local extracellular accumulation of K(+), as indicated by K(+) tail currents (I(tail)) after a depolarization step, is greater in the vicinity of oligodendrocytes than that of astrocytes. It has been suggested that this may reflect a smaller extracellular space (ECS) around oligodendrocytes compared to astrocytes [Chvátal et al. [1997] J. Neurosci. Res. 49:98-106; [1999] J. Neurosci. Res. 56:493-505). We therefore compared the effect of osmotic stress in spinal cord slices from 5-11-day-old rats on the changes in reversal potentials (V(rev)) of I(tail) measured by the whole-cell patch-clamp technique and the changes in ECS volume measured by the real-time iontophoretic method. Cell swelling induced by a 20 min perfusion of hypoosmotic solution (200 mmol/kg) decreased the ECS volume fraction from 0.21 +/- 0.01 to 0.15 +/- 0.02, i.e., by 29%. As calculated from V(rev) of I(tail) using the Nernst equation, a depolarizing prepulse increased [K(+)](e) around astrocytes from 11.0 to 44.7 mM, i.e., by 306%, and around oligodendrocytes from 26.1 to 54.9 mM, i.e., by 110%. The ECS volume fraction decrease had the same time course as the changes in V(rev) of I(tail). Cell shrinkage in hyperosmotic solution (400 mmol/kg) increased ECS volume fraction from 0.24 +/- 0.02 to 0.32 +/- 0.02, i.e., by 33%. It had no effect on [K(+)](e) evoked by a depolarizing prepulse in astrocytes, whereas in oligodendrocytes [K(+)](e) rapidly decreased from 52 to 26 mM, i.e., by 50%. The increase in ECS volume was slower than the changes in [K(+)](e). These data demonstrate that hypoosmotic solution has a larger effect on the ECS volume around astrocytes than around oligodendrocytes and that hyperosmotic solution affects the ECS volume around oligodendrocytes only. This indicates that increased K(+) accumulation in the vicinity of oligodendrocytes could be due to a restricted ECS. Oligodendrocytes in the CNS are therefore most likely surrounded by clusters of "compacted" ECS, which may selectively affect the diffusion of neuroactive substances in specific areas and directions and facilitate spatial K(+) buffering.
Subject(s)
Cell Size/physiology , Extracellular Space/metabolism , Neuroglia/metabolism , Potassium/metabolism , Spinal Cord/metabolism , Stress, Physiological/metabolism , Water-Electrolyte Balance/physiology , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Membrane Permeability/physiology , Electric Stimulation , Membrane Potentials/physiology , Neuroglia/cytology , Oligodendroglia/cytology , Oligodendroglia/metabolism , Organ Culture Techniques , Osmolar Concentration , Osmotic Pressure , Potassium Channels/metabolism , Rats , Spinal Cord/cytology , Stress, Physiological/physiopathologyABSTRACT
Using the patch-clamp technique in the whole-cell configuration combined with intracellular dialysis of the fluorescent dye Lucifer yellow (LY), the membrane properties of cells in slices of the lumbar portion of the frog spinal cord (n=64) and the filum terminale (FT, n=48) have been characterized and correlated with their morphology. Four types of cells were found in lumbar spinal cord and FT with membrane and morphological properties similar to those of cells that were previously identified in the rat spinal cord (Chvátal, A., Pastor, A., Mauch, M., Syková, E., Kettenmann, H., 1995. Distinct populations of identified glial cells in the developing rat spinal cord: Ion channel properties and cell morphology. Eur. J. Neurosci. 7, 129-142). Neurons, in response to a series of symmetrical voltage steps, displayed large repetitive voltage-dependent Na(+) inward currents and K(+) delayed rectifying outward currents. Three distinct types of non-neuronal cells were found. First, cells that exhibited passive symmetrical non-decaying currents were identified as astrocytes. These cells immunostained for GFAP and typically had at least one thick process and a number of fine processes. Second, cells with the characteristic properties of rat spinal cord oligodendrocytes, with passive symmetrical decaying currents and large tail currents after the end of the voltage step. These cells exhibited either long parallel or short hairy processes. Third, cells that expressed small brief inward currents in response to depolarizing steps, delayed rectifier outward currents and small sustained inward currents identical to rat glial precursor cells. Morphologically, they were characterized by round cell bodies with a number of finely branched processes. LY dye-coupling in the frog spinal cord gray matter and FT was observed in neurons and in all glial populations. All four cell types were found in both the spinal cord gray matter and FT. The glia/neuron ratio in the spinal cord was 0.78, while in FT it was 2.0. Moreover, the overall cell density was less in the FT than in the spinal cord. The present study shows that the membrane and morphological properties of glial cells in the frog and rat spinal cords are similar. Such striking phylogenetic similarity suggests a significant contribution from distinct glial cell populations to various spinal cord functions, particularly ionic and volume homeostasis in both mammals and amphibians.
Subject(s)
Cauda Equina/physiology , Membrane Potentials/physiology , Neuroglia/physiology , Neurons/physiology , Rana pipiens/physiology , Spinal Cord/physiology , Animals , Astrocytes/cytology , Astrocytes/physiology , Cauda Equina/cytology , Cell Size/physiology , Electric Stimulation , Fluorescent Dyes/pharmacokinetics , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Isoquinolines/pharmacokinetics , Neuroglia/cytology , Neurons/cytology , Oligodendroglia/cytology , Oligodendroglia/physiology , Patch-Clamp Techniques , Rana pipiens/anatomy & histology , Spinal Cord/cytology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
Although synaptic transmission is an important means of communication between neurons, neurons themselves and neurons and glia also communicate by extrasynaptic "volume" transmission, which is mediated by diffusion in the extracellular space (ECS). The ECS of the central nervous system (CNS) is the microenvironment of neurons and glial cells. The composition and size of ECS change dynamically during neuronal activity as well as during pathological states. Following their release, a number of neuroactive substances, including ions, mediators, metabolites and neurotransmitters, diffuse via the ECS to targets distant from their release sites. Glial cells affect the composition and volume of the ECS and therefore also extracellular diffusion, particularly during development, aging and pathological states such as ischemia, injury, X-irradiation, gliosis, demyelination and often in grafted tissue. Recent studies also indicate that diffusion in the ECS is affected by ECS volume inhomogeneities, which are the result of a more compacted space in certain regions, e.g. in the vicinity of oligodendrocytes. Besides glial cells, the extracellular matrix also changes ECS geometry and forms diffusion barriers, which may also result in diffusion anisotropy. Glial cells therefore play an important role in extrasynaptic transmission, for example in functions such as vigilance, sleep, depression, chronic pain, LTP, LTD, memory formation and other plastic changes in the CNS. In turn, ECS diffusion parameters affect neuron-glia communication, ionic homeostasis and movement and/or accumulation of neuroactive substances in the brain.
Subject(s)
Central Nervous System/physiology , Neuroglia/physiology , Synaptic Transmission/physiology , Aging/physiology , Animals , Central Nervous System/cytology , Diffusion , Extracellular Space/physiologyABSTRACT
The cell membrane of astrocytes and oligodendrocytes is almost exclusively permeable for K+. Depolarizing and hyperpolarizing voltage steps produce in oligodendrocytes, but not in astrocytes, decaying passive currents followed by large tail currents (Itail) after the offset of a voltage jump. The aim of the present study was to characterize the properties of Itail in astrocytes, oligodendrocytes, and their respective precursors in the gray matter of spinal cord slices. Studies were carried out on 5- to 11-day-old rats, using the whole-cell patch clamp technique. The reversal potential (Vrev) of Itail evoked by membrane depolarization was significantly more positive in oligodendrocytes (-31.7+/-2.58 mV, n = 53) than in astrocytes (-57.9+/-2.43 mV, n = 21), oligodendrocyte precursors (-41.2+/-3.44 mV, n = 36), or astrocyte precursors (-52.1+/-1.32 mV, n = 43). Analysis of the Itail (using a variable amplitude and duration of the de- and hyperpolarizing prepulses as well as an analysis of the time constant of the membrane currents during voltage steps) showed that the Itail in oligodendrocytes arise from a larger shift of K+ across their membrane than in other cell types. As calculated from the Nernst equation, changes in Vrev revealed significantly larger accumulation of the extracellular K+ concentration ([K+]e) around oligodendrocytes than around astrocytes. The application of 50 mM K+ or hypotonic solution, used to study the effect of cell swelling on the changes in [K+]e evoked by a depolarizing prepulse, produced in astrocytes an increase in [K+]e of 201% and 239%, respectively. In oligodendrocytes, such increases (22% and 29%) were not found. We conclude that K+ tail currents, evoked by a larger accumulation of K+ in the vicinity of the oligodendrocyte membrane, could result from a smaller extracellular space (ECS) volume around oligodendrocytes than around astrocytes. Thus, in addition to the clearance of K+ from the ECS performed by astrocytes, the presence of the K+ tail currents in oligodendrocytes indicates that they might also contribute to efficient K+ homeostasis.
Subject(s)
Astrocytes/physiology , Neuroglia/physiology , Oligodendroglia/physiology , Potassium/metabolism , Spinal Cord/physiology , Animals , Animals, Newborn , Astrocytes/cytology , Cell Size , Extracellular Space/physiology , In Vitro Techniques , Membrane Potentials , Neuroglia/cytology , Oligodendroglia/cytology , Patch-Clamp Techniques , Rats , Regression Analysis , Spinal Cord/cytology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
The effect of L-glutamate, kainate and N-methyl-D-aspartate (NMDA) on membrane currents of astrocytes, oligodendrocytes and their respective precursors was studied in acute spinal cord slices of rats between the ages of postnatal days 5 and 13 using the whole-cell patch-clamp technique. L-glutamate (10(-3) M), kainate (10(-3) M), and NMDA (2x10(-3) M) evoked inward currents in all glial cells. Kainate evoked larger currents in precursors than in astrocytes and oligodendrocytes, while NMDA induced larger currents in astrocytes and oligodendrocytes than in precursors. Kainate-evoked currents were blocked by the AMPA/kainate receptor antagonist CNQX (10(-4) M) and were, with the exception of the precursors, larger in dorsal than in ventral horns, as were NMDA-evoked currents. Currents evoked by NMDA were unaffected by CNQX and, in contrast to those seen in neurones, were not sensitive to Mg2+. In addition, they significantly decreased during development and were present when synaptic transmission was blocked in a Ca2+-free solution. NMDA-evoked currents were not abolished during the block of K+ inward currents in glial cells by Ba2+; thus they are unlikely to be mediated by an increase in extracellular K+ during neuronal activity. We provide evidence that spinal cord glial cells are sensitive to the application of L-glutamate, kainate and transiently, during postnatal development, to NMDA.
Subject(s)
Glutamic Acid/pharmacology , Ion Channels/drug effects , Kainic Acid/pharmacology , N-Methylaspartate/pharmacology , Neuroglia/physiology , Spinal Cord/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Astrocytes/physiology , Astrocytes/ultrastructure , Cell Membrane/drug effects , Cell Membrane/physiology , Electric Conductivity , Excitatory Amino Acid Antagonists/pharmacology , Ion Channels/physiology , Membrane Potentials , Neuroglia/ultrastructure , Oligodendroglia/physiology , Oligodendroglia/ultrastructure , Patch-Clamp Techniques , Rats , Receptors, AMPA/antagonists & inhibitors , Receptors, Kainic Acid/antagonists & inhibitors , Spinal Cord/ultrastructureABSTRACT
Whole cell patch-clamp recordings of K+ currents from oligodendrocyte precursors in 10-day-old rats (P10) and, following myelination, in mature oligodendrocytes from 20-day-old rats (P20) were correlated with extracellular space (ECS) diffusion parameters measured by the local diffusion of iontophoretically injected tetramethylammonium ions (TMA+). The aim of this study was to find an explanation for the changes in glial currents that occur with myelination. Oligodendrocyte precursors (P10) in slices from corpus callosum were characterized by the presence of A-type K+ currents, delayed and inward rectifier currents, and lack of tail currents after the offset of a voltage jump. Mature oligodendrocytes in corpus callosum slices from P20 rats were characterized by passive, decaying currents and large tail currents after the offset of a voltage jump. Measurements of the reversal potential for the tail currents indicate that they result from increases in [K+]e by an average of 32 mM during a 20 msec 100 mV voltage step. Concomitant with the change in oligodendrocyte electrophysiological behavior after myelination there is a decrease in the ECS of the corpus callosum. ECS volume decreases from 36% (P9-10) to 25% (P20-21) of total tissue volume. ECS tortuosity lambda = (D/ADC)0.5, where D is the free diffusion coefficient and ADC is the apparent diffusion coefficient of TMA+ in the brain, increases as measured perpendicular to the axons from 1.53 +/- 0.02 (n = 6, mean +/- SEM) to 1.70 +/- 0.02 (n = 6). TMA+ non-specific uptake (k') was significantly larger at P20 (5.2 +/- 0.6 x 10(-3) s(-1), n = 6) than at P10 (3.5 +/- 0.4 x 10(-3) s(-1), n = 6). It can be concluded that membrane potential changes in mature oligodendrocytes are accompanied by rapid changes in the K+ gradient resulting from K+ fluxes across the glial membrane. As a result of the reduced extracellular volume and increased tortuosity, the membrane fluxes produce larger changes in [K+]e in the more mature myelinated corpus callosum than before myelination. These conclusions also account for differences between membrane currents in cells in slices compared to those in tissue culture where the ECS is essentially infinite. The size and geometry of the ECS influence the membrane current patterns of glial cells and may have consequences for the role of glial cells in spatial buffering.
Subject(s)
Corpus Callosum/metabolism , Extracellular Space , Ion Channel Gating , Oligodendroglia/metabolism , Potassium Channels/physiology , Potassium/metabolism , Animals , Corpus Callosum/cytology , Corpus Callosum/growth & development , Diffusion , Membrane Potentials , Mice , Myelin Sheath/physiology , Patch-Clamp Techniques , Rats , Rats, Wistar , Species SpecificityABSTRACT
In the neonatal rat spinal cord, four types of glial cells, namely astrocytes, oligodendrocytes and two types of precursor cells, can be distinguished based on their membrane current patterns and distinct morphological features. In the present study, we demonstrate that these cells respond to the inhibitory neurotransmitters glycine and GABA, as revealed with the whole-cell recording configuration of the patch-clamp technique. All astrocytes and glial precursor cells and a subpopulation of oligodendrocytes responded to glycine. The involvement of glycine receptors was inferred from the observation that the response was blocked by strychnine and that the induced current reversed close to the Cl- equilibrium potential. GABA induced large membrane currents in astrocytes and precursor cells while oligodendrocytes showed only small responses. The GABA-activated current was due to the activation of GABAA receptors since muscimol mimicked and bicuculline blocked the response; moreover, the reversal potential was close to the Cl- equilibrium potential. Besides the increase in a Cl- conductance, GABAA receptor activation also induced a block of the resting K+ conductance, as observed previously in Bergmann glial cells. Our experiments show that while glial GABAA receptors are found in many brain regions and the spinal cord, glial glycine receptors have so far been detected only in the spinal cord. The restricted coexpression of glial and neuronal glycine receptors in a defined central nervous system grey matter area implies that such glial receptors may be involved in synaptic transmission.